Chromosomal translocations involving the mixed lineage leukemia (MLL) gene occur frequently in human acute leukemias of myeloid and lymphoid lineages. We identified the Set1 protein of yeast Saccharomyces cerevisiae as a MLL homologue and purified it in a complex we call COMPASS. Set1/COMPASS is capable of methylating histone H3 on its K4 (H3K4). Based on the yeast studies, we now know that human MLL is also found in a COMPASS-like complex capable of methylating H3K4. The yeast studies in our laboratory during the past ten years have resulted in the identification of the molecular machineries involved in histone H3K4 methylation by COMPASS and H3K79 methylation by Dot1. For example, we demonstrated that histone H2B monoubiquitination by Rad6/Bre1 is required for proper H3K4 trimethylations by COMPASS and Dot1. These enzymatic machineries identified in yeast are highly conserved from yeast to human. Given the fact that human MLL and Dot1 are involved in the pathogenesis of leukemia, the central hypothesis of this study is that information obtained from studies in yeast in this regard will have a direct and valuable impact on our understanding and the treatment of MLL translocation-based leukemia. These objectives will be achieved through three specific aims. We have recently been able to fully reconstitute active yeast COMPASS. Therefore the Specific Aim 1 of this application will be focused on defining how each subunit of the complex contributes to the process of H3K4 methylation; and how H2BK123 monoubiquitination alters the catalytic properties of the enzyme. Our recent molecular studies demonstrated that a surprisingly small amount of H2B monoubiquitination is enough to provide almost a full level of H3K4 trimethylation in yeast cells. Given that we have recently developed H2B monoubiquitinated specific polyclonal antibodies, the Specific Aim 2 of this application is focused on identifying factors required for proper H2B monoubiquitination independently of H3K4 methylation by employing genetic and biochemical screens. Our studies in yeast have demonstrated that histone H3K79 methylation is a dynamic process involved in transcriptional regulation. However, there are no known H3K79 demethylases. Therefore, Specific Aim 3 of the application is focused on the use of molecular screens identifying H3K79 demethylase machinery in yeast S. cerevisiae and full molecular and biochemical characterization of these factors. Data obtained as the result of the implementation of the above proposed three aims will not only have a fundamental impact on our understanding of the regulation of histones H2BK123 by monoubiquitination and H3K4/K79 by methylations, but also will be instrumental in obtaining a comprehensive understanding of the roles these factors play in the pathogenesis of MLL translocation-based hematological malignancies, and how such pathways could be used for targeted therapeutics for leukemia caused by MLL translocations. PUBLIC HEALTH RELEVANCE: The focus of this renewal application is on defining the molecular machineries and mechanisms involved in the implementation and removal of histones H2B monoubiquitination and H3K4 and H3K79 methylations in yeast Saccharomyces cerevisiae. In human, these marks and the machineries are associated with the pathogenesis of childhood leukemia. We plan to characterize the biochemical, molecular, and enzymatic properties of these factors in yeast and to generate small molecule inhibitors for their activities with the hope that they can be used for targeted therapeutics for translocation-based leukemia.